ETHYLENE BIOSYNTHESIS Jean-Claude PECH

Until 1964, ethylene was though to derive from ETHYLENE ORIGINATING FROM LIPIDS? Until 1964, ethylene was though to derive from the enzymatic disruption of lipids involving free radicals formation Arguments: -Oxygen is required for the biosynthesis of ethylene in plants; -Free radical scavengers (antioxidants that prevent lipid oxidation, e.g. propyl gallate ...) inhibited ethylene production.

DISCOVERY OF METHIONINE AS A PRECURSOR OF ETHYLENE * * Methionine NH3+ CH3-S-CH2-CH2-CH-COO- DISCOVERY OF METHIONINE AS A PRECURSOR OF ETHYLENE Lieberman and Mapson, 1964

OTHER INHIBITORS OF ETHYLENE SYNTHESIS Lieberman and Owens, 1971 CH2-CH-CH2-O-C=C-CH-COOH OH NH2 H NH2 H Rhizobitoxine from Rhizobium japonicum CH2-CH2-CH2-O-C=C-CH-COOH NH2 H NH2 H AVG= Amino ethoxy vinyl glycine from Streptomyces

Shang-Fa YANG DISCOVERY OF ACC AND METHIONINE CYCLE « Knight of Roquetaillade », Ethylene Congress Agen, 1992

Radio-chromatograms of ethylene extracts of apple plugs 14C-Methionine Radio-chromatograms of ethylene extracts of apple plugs Plugs in air, 12h Plugs in N2, 6h Plugs in N2, 6h then in air, 6h

? ? * * * * * * Methionine NH3+ CH3-S-CH2-CH2-CH-COO- * * Methionine NH3+ CH3-S-CH2-CH2-CH-COO- S-Adenosyl Methionine * * NH3+ CH3-S-CH2-CH2-CH-COO- Ade + 1-aminocyclopropane-1 -carboxylic acid CH2 CH2 CH NH3+ COO- * ? ? CH2 CH2 * Ethylene

Ethylene biosynthesis 2-keto-4-methylthiobutyric acid (KMBA) transamination Pi+HOOCH O2 L-methionine 5’-methylethioribose-1-phosphate (MTR-1-P) Yang Cycle ATP+H2O Pi+PPi ATP ADP S-adenosyl-methionine (AdoMet) 5’-methylthioribose (MTR) H2O adenine 5’-methylthioadenosine (MTA) H2C NH3 C H2C COO- ACC 1/2 O2 HCN+H2O+CO2 C=C H Ethylene

Abstract

« Knight of Roquetaillade », Ethylene Congress Agen, 1992 Don GRIERSON, DISCOVERY OF ACC OXIDASE « Knight of Roquetaillade », Ethylene Congress Agen, 1992

DISCOVERY OF A METHOD FOR MEASURING EFE IN A SOLUBLE FORM Ververidis and John, 1991: Complete recovery in vitro of ethylene forming enzyme activity. Phytochemistry, 30:725-727

ACC SYNTHASE PURIFICATION FROM WOUNDED ZUCCHINI Unpurified serum Purified serum Silver staining Pre-immune 1: Highly impure extract 2: Highly pure extract

Ethylene biosynthesis 2-keto-4-methylthiobutyric acid (KMBA) transamination Pi+HOOCH O2 L-methionine 5’-methylethioribose-1-phosphate (MTR-1-P) Yang Cycle ATP+H2O Pi+PPi ATP ADP S-adenosyl-methionine (AdoMet) 5’-methylthioribose (MTR) H2O adenine 5’-methylthioadenosine (MTA) ACC synthase H2C NH3 C H2C COO- ACC GACC MACC 1/2 O2 ACC oxidase HCN+H2O+CO2 C=C H Ethylene

Inhibitors of ACC synthase: Rhizobitoxine, AVG EFFECTORS OF ACC SYNTHASE AND ACC OXIDASE Inhibitors of ACC synthase: Rhizobitoxine, AVG Factors stimulating ACS gene expression: Auxins, chilling, wounding, drought, pathogens, ethylene Inhibitors of ACC oxidase: Anoxia, uncouplers, free radical scavengers, Co++, Ni++, T°>35°C, Factors stimulating ACO gene expression: Pathogens, ethylene, wounding

ACC SYNTHASE GENE FAMILY IN ARABIDOPSIS At-ACS5 [induced by Li, cycloheximide(CH) and low concentration of cytokinin(CK)] At-ACS4 (induced by CH, IAA and wounding) At-ACS1 At-ACS2 Le-ACS2 Le-ACS4 At-ACS6 Le-ACS6 Le-ACS1A Le-ACS1B At-ACS7 At-ACS11 Le-ACS5 At-ACS4 At-ACS8 At-ACS5 At-ACS9 Le-ACS7 Le-ACS3 Le-ACS8 At-ACS10 At-ACS12 Branch I Branch II Branch III * At-ACS1 (induced by cycloheximide) At-ACS10 in response to light in plants over expressing CONSTANS At-ACS8 not yet characterized At-ACS2 in young leaves and flowers At-ACS6 (induced by O3, Cu2+, IAA, CH, CN-, anaerobiosis, wounding, touching and ethylene) At-ACS4 (induced by CH, IAA, anaerobisis and wounding) At-ACS7 (induced by CH) At-ACS5 (basal level stimulated by Li and CK At-ACS2 (induced by Li, CH, wounding) At-ACS4 induced by CH, IAA, wounding) At-ACS9 = eto1 Adult plant Etiolated seedling

ACO AND ACS GENE EXPRESSION IN TOMATO FLOWER Le-ACS2 (stamens, mature and senescent anthers) Le-ACO2 Le-ACS2 (senescent petals) Le-ACO4 Le-ACO1, 3 Le-ACS3 (not expressed in fruit, present in tomato cell suspension) Le-ACO3 (breaker stage) Le-ACS4 induced by wounding Le-ACO4 Le-ACS2 and 4 positiv. regul. by ethylene Le-ACO1,2,3 and 4 induced by O3 Le-ACS1A (breaker stage) negativ. regul. by ethylene Le-ACS6 (in MG fruit) negativ. regul. by ethylene Le-ACS7 induced early by wounding Le-ACO1 in senescent or wounded leaves Le-ACS3 induced by flooding Le-ACS7 induced early by flooding

Regulation of ethylene biosynthetic genes ACC synthase Arabidopsis Rapidly induced by auxin (ACS4). Induced by low levels of cytokinin (ACS5). Rumex Flood resistant species downregulates ACS, flood sensitive does not. Rice Enzyme activity induced by hypoxia/flooding. Mung bean Induced by mechanical stress (AIM1). Tomato Induced by fruit ripening. Induced by flooding (ACS3). Induced by ozone (ACS2). Phaseolus Induced by auxin (ACS1). Synergistic induction with auxin (ACS2). Winter squash Induced by auxin (accA). Induced by wounding (accW). Phalaenopsis Induced by ethylene (ACS1) Induced in stigma after pollination or auxin treatment (ACS1, ACS2). Induced in ovary after pollination or auxin treatment (ACS3). Carnation Late induction by ethylene, pollination induces expression after 12-24 hours in petals (CARACC3). Stellaria Regulated by photoperiod. Potato Rapidly induced by ozone, infection and Cu2+ (ACS5). Slowly induced by ozone, infection and Cu2+ (ACS4). Zucchini Induced by wounding and auxin only in fruit (ACC1A). ACC oxidase Arabidopsis Induced by ethylene (ACO2/E1305). Tomato ACO1 antisense transgenic plants have reduced ethylene production and epinasty after flooding. Induced during flower development (ACO1-3). Induced by wounding (ACO1). Induced during senescence (ACO, ACO3). Phalaenopsis Induced by ethylene. Unpollinated flowers cannot convert ACC to ethylene. Cantaloupe Induced by ethylene, wounding, NaCl, drought and ripening (ACO1). Induced by NaCl and drought (ACO3). Petunia Induced by ethylene (ACO3). Induced in senescing floral organs (ACO1, ACO3). Induced in developing pistils (ACO3, ACO4). Winter Squash Induced by wounding, induction blocked by ethylene inhibitor, endogenous ethylene accelerates induction. Carnation Induced by pollination in styles and petals (SR120). Mung bean Induced by ethylene(ACO1, ACO2). Peach Induced by wounding and fruit ripening. Apple Induced during fruit ripening. Broccoli Induced after harvest (ACC Ox1). Induced in reproductive organs postharvest (ACC Ox2).